EP2421994B1

EP2421994B1 - Bin1 as a prognostic marker in cardiovascular disease

Info

Publication number: EP2421994B1
Application number: EP10767856.7A
Authority: EP
Inventors: Robin Shaw; Ting-ting HONG; James Smyth
Original assignee: University of California
Current assignee: University of California
Priority date: 2009-04-24
Filing date: 2010-04-23
Publication date: 2015-08-05
Anticipated expiration: 2030-04-23
Also published as: EP2421994A2; US20120094300A1; CA2759698C; EP2421994A4; US20140302502A1; WO2010124240A2; JP2012524550A; US9150924B2; CA2759698A1; JP5802197B2; WO2010124240A3

Description

This invention relates to cardiovascular disease and methods to assess the same.
Daizell et al, Biomarkers Med., (2009) 3(5); 453-463 provide an overview of novel biomarkers in heart failure. Muller et al, Mol. Cell. Biol., (2003), 23; 4295-4306, report on the effect of targeted disruption of the murine Bin1 gene.
During the last decade, congestive heart failure (CHF) has burgeoned into the most prominent public health problem in cardiovascular medicine. CHF affects close to 20 million people worldwide and approximately 5 million Americans. In the United States alone, about 400,000 new cases of CHF are diagnosed annually and the condition is responsible for approximately 200,000 deaths per year. About a 100,000 patients diagnosed with CHF have end-stage heart failure. Currently, the treatment for these patients with end-stage heart failure is heart transplant. However, only 2000 hearts are available each year for heart transplant. Mechanical assist devices may be used to stabilize patients waiting for a heart transplant. In some cases, mechanical assist devices are being considered as a treatment option rather than a bridge to heart transplant.
There is a need for diagnostic methods for diagnosing cardiac health, and for evaluating treatment options and efficacy of treatment.

SUMMARY

The present disclosure provides methods involving use of BIN 1 expression levels in evaluating the risk of a poor outcome in a patient diagnosed with congestive heart failure. The methods find use in evaluating patients who are heart transplant candidates, as well as in determining therapy options and assessing efficacy of treatment in congestive heart failure patients.
Accordingly, the present disclosure provides a method as defined in claim 1 for determining a risk of a poor outcome in a congestive heart failure patient. Poor outcome can include, for example, cardiac mortality, the need for mechanical device support (e.g., a left ventricular assist device (LVAD) implant), or a left ventricular ejection fraction of less than 25%. In certain embodiments, the congestive heart failure patient has received a heart transplant and the heart tissue sample is from the transplanted heart. In related embodiments, the heart of the congestive heart failure patient is connected to a mechanical assist device. In other embodiments, the patient is undergoing treatment for CHF. In further embodiments, the patient is undergoing immunosuppression therapy. A decreased BIN1 expression level compared to a reference BIN expression level is positively correlated to an increase in the risk of a poor outcome.
In exemplary embodiments, the assaying comprises measuring the level of BIN1 mRNA. In certain cases, the assaying comprises measuring the level of BIN1 protein. In yet other embodiments, the method comprises assaying a CaV1.2 expression level and normalizing the BIN1 expression level using CaV1.2 expression level.
The present disclosure also provides a method as defined in claim 6 for determining a risk of a poor outcome in a congestive heart failure patient.
In exemplary embodiments, the internal control gene is a housekeeping gene. In other examples, the internal control gene is CaV1.2. In certain embodiments, the assaying comprises measuring the levels of BIN1 and the internal control gene mRNA. In related embodiments, the congestive heart failure patient has received a heart transplant and the heart tissue sample is from the transplanted heart. In alternate embodiments, the heart of the congestive heart failure patient is connected to a mechanical assist device. In related embodiments, the patient is undergoing treatment for CHF. In certain embodiments, the patient is undergoing immunosuppression therapy. In certain embodiments, the IDF ratio of the patient is compared to a reference IDF and an IDF ratio greater than than the reference IDF ratio is indicative of an increased risk of a poor outcome

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 depicts CaV1.2 expression in human heart. (A) Quantitative RT-PCR analysis (left) and semi-quantitative western blotting show no difference in CaV 1.2 expression in both non-failing and failing human hearts. (B) Three dimensional volume view of intracellular CaV1.2 distribution reconstructed from a stack of 100x confocal image frames acquired at a z-step of 0.1µum.
Figure 2 shows BIN1 mediated targeting of CaV1.2 to T-Tubules. (A) Electron microscope image of a mouse cardiomyocyte fixed and immunogold labeled BIN1 (small dots at of 10nm) and CaV1.2 (large dots at ∅ 15nm) (Scale bar: 200nm). BIN1 and CaV1.2 cluster within 50nm on T-tubule membranes close to Z-line. (B) CaV1.2 immunoprecipitates with BIN1.
Figure 3 shows CaV1.2 targeting requires BIN1 not T-tubule structure. (A) Domain map of wild-type BIN 1 (BIN1-WT) shows a N-terminal BAR domain followed by 15 amino acids encoded by exon 10, the coiled-coil region and the C-terminal SH3 domain. (B) Surface biotinylation of CaV1.2 in HL-1 cells transfected with either BIN1-WT or a BIN1 truncation (BIN1-BAR* which retains the T-tubule forming domain but lacks CaV1.2 binding domain) shows that whole length BIN1 is required to induce surface expression of CaV1.2.
Figure 4 depicts expression levels of BIN1 in heart and T-tubule morphology. (A) Quantitative rt-PCR analysis (left) and semi-quantitative western blotting show significant reduction of BIN1 expression in failing human hearts. (B) Confocal image (100x) of isolated human cardiomyocytes stained with BIN1 indicate much less expression of BIN1 and much shallower T-tubule structures defined by BIN1 staining. 3D volume views of WGA-labeled human cardiomyocytes reconstructed from stacks of 100x confocal image frames acquired at a z-step of 0.1µm reveal a loss of organized T-tubule network in failing human cardiomyocytes.
Figure 5 depicts microtubule dependent forward trafficking of CaV1.2. Surface biotinylation of mouse cardiomyocytes showed that nocodazole reduced surface CaV1.2 expression, indicating a need for microtubules in CaV1.2 trafficking.
Figure 6 shows CaV1.2 targeting involves APC-tipped microtubules (A) Surface biotinylation shows dominant negative APC (DN APC) reduced surface CaV1.2 expression in HL-1 cells. (B) APC particles travel a longer distance at a faster velocity when BIN1 is absent.
Figure 7 depicts BIN1 expression levels in non-failing normal heart (A) and in failing heart (B). (A) Non-failing heart from individuals who died for reasons other than heart disease exhibit comparable BIN1 expression levels in left ventricle free wall (LVFW) and LV Apex. (B) Hearts from end stage dilated cardiomyopathy patients who received LVAD at LV Apex show a recovery of BIN1 expression in the LV apex heart tissue (compare to LV-FW).
Figure 8 depicts Intrinsic Disease Factor (IDF_CaV, measured as a ratio of CaV1.2 mRNA to BIN1 mRNA) for normal and diseased heart. (A) Cardiac IDF_CaV for explanted donor hearts. IDF_CaV for non-failing heart from individuals who died for reasons other than heart disease (filled circles) and for failing hearts from end staged dilated cardiomyopathy patients (filled squares). Cardiac IDF_CaV is significantly higher in hearts with end-stage dilated cardiomyopathy. (B) Cardiac IDF_CaV correlates with left ventricular ejection fraction (LVEF). IDF_CaV for non-failing heart from normal control individuals who died for reasons other than heart disease (filled squares) and for failing hearts from end-stage dilated cardiomyopathy patients (filled circles).
Figure 9 depicts Intrinsic Disease Factor (IDF_HK, measured as a ratio of a housekeeping gene mRNA to BIN1 mRNA) for normal and diseased heart. (A) Cardiac IDF (IDF_HK, measured as a ratio of a HPRT1 mRNA to BIN1 mRNA) for explanted donor hearts. IDF_HK for non-failing heart from normal control individuals who died for reasons other than heart disease (filled circles) and for failing hearts from end-stage dilated cardiomyopathy patients (filled squares). Cardiac IDF_HK is significantly higher in hearts with end-stage dilated cardiomyopathy. (B) Cardiac IDF_HK correlated to left ventricular ejection fraction (LVEF). IDF_HK for non-failing heart from normal control individuals who died for reasons other than heart disease (filled squares) and for failing hearts from end-stage dilated cardiomyopathy patients (filled circles).
Figure 10 illustrates the correlation of BIN expression level (normalized to CaV1.2) with cardiac output. The ratio of BIN1 to Cav1.2 is the reciprocal of IDF_CaV.
Figures 11A and 11B provide exemplary BIN1 nucleotide (transcript variant 8) (SEQ ID NO: 1) and amino acid (isoform 8) (SEQ ID NO: 2) sequences, respectively.
Figures 12A and 12B provide exemplary BIN1 nucleotide (transcript variant 9) (SEQ ID NO: 3) and amino acid (isoform 9) (SEQ ID NO: 4) sequences, respectively.
Figures 13A and 13B provide exemplary CaV1.2 nucleotide (SEQ ID NO: 5) and amino acid (SEQ ID NO: 6) sequences, respectively.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

Before the present invention is described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and exemplary methods and materials are now described. All publications mentioned herein disclose and describe the methods and/or materials in connection with which the publications are cited. It is understood that the present disclosure supersedes any disclosure of an incorporated publication to the extent there is a contradiction.
It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a housekeeping gene" includes a plurality of such genes and reference to "the gene" includes reference to one or more genes and equivalents thereof known to those skilled in the art, and so forth.
It is further noted that the claims may be drafted to exclude any element which may be optional. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely", "only" and the like in connection with the recitation of claim elements, or the use of a "negative" limitation.
The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

Definitions

The terms "heart failure" or "congestive heart failure" (CHF), and "congestive cardiac failure" (CCF), are used interchangeably herein, and refer to a clinical condition that may result from any structural or functional cardiac disorder that impairs the ability of the heart to fill with or pump a sufficient amount of blood throughout the body to maintain adequate circulation of blood in the tissues of the body or to pump out the venous blood returned to it by the venous circulation.
The term "End-stage heart failure" refers to CHF that is refractory to conventional medical therapy. Patients with end-stage heart failure have a high mortality rate. These patients frequently undergo multiple frequent hospitalizations, intravenous medications, and require surgical therapies such intraaortic balloon pumps, ventricular assist devices, and heart transplant.
The terms "end-stage dilated cardiomyopathy" and "end-stage CHF" are used interchangeably herein.
The term "cardiomyopathy" or "heart muscle disease" refers to the deterioration of the function of the myocardium (i.e., the heart muscle) for any reason. As used herein, the term "cardiomyopathy" includes "extrinsic cardiomyopathies" and "intrinsic cardiomyopathies". In extrinsic cardiomyopathies the primary pathology is outside the myocardium itself, for example, ischemic cardiomyopathy. In intrinsic cardiomyopathies, weakness in the heart muscle is not due to an identifiable external cause, for example, Dilated cardiomyopathy (DCM). In DCM the heart (especially the left ventricle) is enlarged and the pumping function is diminished.
The term "Ischemic cardiomyopathy" refers to cardiomyopathy that results from coronary artery disease, such as atherosclerosis and occlusion of the coronary arteries.
The term "Non-ischemic cardiomyopathy" refers to cardiomyopathy that is not due to coronary artery disease.
The term "prognosis" as used herein refers to a prediction of likelihood of a particular outcome of a disease in a patient, such as likelihood of cardiac mortality in a cardiac disease patient, such as a CHF patient.
"Poor outcome" as used herein in the context of a cardiac patient (e.g., a heart failure patient) refers to an outcome associated with declining heart health. "Poor outcome" includes, for example, in a given time period, a failure of left ventricular ejection fraction (LVEF) to improve or the need for mechanical device support (e.g., a left ventricular assist device (LVAD) implant), or the need for a heart transplant, or cardiac death.
The term "treating" or "treatment" of a condition or disease includes providing a clinical benefit to a subject, and includes: (1) inhibiting the disease, i.e., arresting or reducing the development of the disease or its symptoms, or (2) relieving the disease, i.e., causing regression of the disease or its clinical symptoms.

Overview

The methods of the present disclosure are based on the discovery that decreasing BIN1 expression levels are positively correlated with declining health of cardiac tissue, and thus with an increasing likelihood of a poor outcome in a cardiac disease patient having congestive heart failure (CHF). For example, as BIN1 expression levels in heart tissue decrease relative to a reference BIN1 expression level, such as a normal BIN1 expression level of normal heart tissue or BIN1 expression level from heart tissue of a patient with congestive heart failure who did not show a poor outcome, the risk of cardiac mortality in the patient increases. The term "cardiac mortality", as used herein, refers to patient mortality due to cardiac disease. Accordingly, BIN1 expression can be used as a prognostic marker that is assayed to provide an expression level value that decreases as the health of heart tissue decreases.
The methods disclosed herein also provide for use of a BIN1 expression level to calculate an Intrinsic Disease Factor (IDF), which provides a score that increases in value as heart tissue health declines. IDF is calculated as a ratio of control gene(s) expression to BIN1 expression. Accordingly, IDF is represented by the formula: $IDF = [Control / BIN 1]$

wherein "Control" represents an expression value for one or more internal control genes, i.e., a gene(s) which is not significantly differentially expressed in diseased versus normal human heart tissue. Exemplary internal control genes include CaV 1.2, and/or housekeeping genes. Accordingly, increasing IDF values are positively correlated with increasing risk of a poor outcome, i.e., the higher the IDF, the greater the risk of poor outcome (e.g., the greater the risk of, for example, cardiac mortality, requirement for a LVAD implant, a failure for LVEF to improve from severe dysfunction..
BIN1 expression levels can be assayed by detection of a BIN1 gene product, e.g., by detection of mRNA (e.g., by detection of cDNA generated from mRNA in a heart tissue sample) or BIN1 protein. Exemplary methods for assaying BIN1 expression are provided below.
The methods of the present disclosure are described in further detail below.

BIN1

Bridging integrator 1 (BIN1) gene encodes a nucleocytosolic protein which was initially identified as a Myc-interacting protein with features of a tumor suppressor. BIN1 is also known as amphiphysin II, amphiphysin-like, and box dependant MYC interacting protein 1. Alternate splicing of the BIN1 gene results in ten transcript variants encoding different isoforms. Some isoforms of BIN1 are expressed ubiquitously while others show a tissue specific expression. BIN1 isoforms 1-7 are expressed in neurons. Isoform 8 is muscle specific while isoforms 9 and 10 are ubiquitous. Isoforms that are expressed in the central nervous system may be involved in synaptic vesicle endocytosis and may interact with dynanim, synaptojanin, endophilin, and clathrin. Aberrant splice variants expressed in tumor cell lines have also been described.
BIN1 expression can be assayed by detection of one or more of the BIN1 transcript variants and/or isoforms. BIN1 isoform or transcript variant 1 mRNA (NM_139343.1) and isoform 1 protein (NP_647593.1), BIN1 transcript variant 2 mRNA (NM_139344.1) and isoform 2 protein (NP_647594.1), BIN1 transcript variant 3 mRNA (NM_139345.1) and isoform 3 protein (NP_647595.1), BIN1 transcript variant 4 mRNA (NM_139346.1) and isoform 4 protein (NP_647596.1), BIN1transcript variant 5 mRNA (NM_139347.1) and isoform 5 protein (NP_647597.1), BIN1 variant 6 mRNA (NM_139348.1) and isoform 6 protein (NP_647598.1), BIN1 transcript variant 7 mRNA (NM_139349.1) and isoform 7 protein (NP_647599.1), BIN1 transcript variant 8 mRNA (NM_004305.2) and isoform 8 protein (NP_004296.1), BIN1 transcript variant 9 mRNA (NM_139350.1) and isoform 9 protein (NP_647600.1), and BIN1 transcript variant 10 mRNA (NM_139351.1) and isoform 10 protein (NP_647601.1) sequences are available in the art. In certain embodiments, BIN1 expression may be assayed by detection of BIN1 transcript variant 8 mRNA and/or BIN1 isoform 8 protein. In other embodiments, BIN1 expression may be assayed by detection of BIN1 transcript variant 9 mRNA and/or BIN1 isoform 9 protein. In other embodiments, BIN1 expression is assayed by detection of both BIN1 transcript variant 8 mRNA and BIN1 transcript variant 9 mRNA and/or detection of both BIN1 isoform 8 protein and BIN1 isoform 9 protein. In exemplary embodiments, BIN1 expression may be assayed by detection of BIN1 transcript variant 8 and 9 mRNA, for example, by detection of structural features shared by BIN1 transcript variants 8 and 9 mRNA and/or by detection of structural features shared by BIN1 isoform 8 protein and BIN1 isoform 9 protein.
In general, BIN1 expression levels may be assayed by using reagents that provide for detection of structure features shared by gene products of the various BIN1 transcript variants/isoforms, e.g., by using primers and/or probes that provide for detection of a region of BIN1 mRNA common to all transcript variants of BIN1 or an antibody that binds an epitope(s) shared by BIN1 isoforms.

INTERNAL CONTROLS

A variety of different internal controls can be used in connection with the assay methods described herein. In general, a gene that is known not to be significantly differentially expressed in heart tissue of a CHF patient and normal heart tissue may be used as an internal control. The following provides exemplary internal control genes which can be assayed.

CaV 1.2

CACNA1C encodes Cav1.2, an alpha-1 subunit of a voltage-dependent calcium channel. Amino acid sequence of Homo sapiens CACNAlc polypeptides are known in the art. See, e.g., GenBank Accession No. NP_000710. Amino acid sequences of Homo sapiens CACNA1C isoforms CRA_a through CRA_p are found under GenBank Accession Nos. EAW88895 (isoform CRA_a); EAW88896 (isoform CRA_b); EAW88897 (isoform CRA_c); EAW88898 (isoform CRA_d); EAW88899 (isoform CRA_e); EAW88900 (isoform CRA_f); EAW888901 (isoform CRA_g); EAW888902 (isoform CRA_h); EAW888903 (isoform CRA_i); EAW888904 (isoform CRA_j); EAW888905 (isoform CRA_k); EAW888906 (isoform CRA_l); EAW888907 (isoform CRA_m); EAW888908 (isoform CRA_n); EAW888909 (isoform CRA_o); and EAW888910 (isoform CRA_p). Corresponding nucleotide sequences (e.g., nucleotide sequences encoding CaV1.2) are known in the art. See, e.g,. GenBank Accession No. NM_000719 (encoding CaV1.2 having the amino acid sequence provided in GenBank Accession No. NP_000710).
In general, CaV1.2 expression levels may be assayed by using reagents that provide for detection of structure features shared by gene products of the various CaV1.2 transcript variants/isoforms, e.g., by using primers and/or probes that provide for detection of a region of CaV1.2 mRNA common to all transcript variants of CaV1.2 or an antibody that binds an epitope(s) shared by CaV1.2 isoforms. In certain embodiments, CaV1.2 transcript variant 18 (NM_000719.6) may be assayed.
Cav1.2 expression level may be assayed by detection of CaV1.2 mRNA or protein.

Housekeeping Genes

A housekeeping gene is typically a constitutively expressed gene that is transcribed at a relatively constant level in a tissue of interest across conditions being evaluated herein. The housekeeping genes typically encode gene products that facilitate maintenance of cells, such nucleic acid synthesis, metabolism, cytoskeletal structure, and the like. In general, expression level of a housekeeping gene is not substantially differentially expressed between a failing and a non-failing heart tissue. Examples of housekeeping genes include HPRT, GAPDH, β-actin, tubulin, ubiquitin, RPL13A, PPIA, and EEF1A1 and the like.
Housekeeping gene expression level may be assayed by detection of mRNA or protein.

METHODS FOR ASSAYING GENE EXPRESSION

Gene expression may be assayed by quantifying the levels of BIN1 mRNA or polypeptide. Commonly used methods known in the art for the quantification of mRNA expression in a sample may be employed for assaying BIN1 expression levels. Such methods include PCR-based methods, such as reverse transcription polymerase chain reaction (RT-PCR) (Weis et al., Trends in Genetics 8:263-264 (1992)), in situ hybridization (Parker & Barnes, Methods in Molecular Biology 106:247-283 (1999)), northern blotting, and RNAse protection assays (Hod, Biotechniques 13:852-854 (1992)). Alternatively, antibodies may be employed that can recognize sequence-specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. The levels of BIN1 polypeptide may be detected by quantitative western-blot, immunoprecipitation, etc. In certain cases, BIN-1 expression levels may be determined by in situ assays, for example, by hybridizing labeled probes specific to BIN-1 mRNA or using a BIN-1 antibody.
In general, BIN1 gene expression may be assayed in a sample of heart tissue from a patient. The heart tissue is usually a biopsy sample. The heart tissue may be biopsied in a variety of procedures, well known to one of skill in the art. The heart tissue may be processed by any method compatible with the assay to be conducted. For example, the heart tissue may be freshly obtained, frozen (e.g., flash frozen), formalin fixed, paraffin embedded, or any suitable combination thereof. In general, the method may be selected so as to avoid substantial degradation of the analyte to be assayed (e.g., proteins and/or mRNA) in the heart tissue.

Quantitative Reverse Transcriptase PCR (qRT-PCR)

The first step is the isolation of mRNA from a sample. The starting material is typically total RNA isolated from the heart tissue of a patient diagnosed with congestive heart failure and, optionally, from corresponding normal tissue as a control. Heart tissue may be obtained fresh or may be frozen or archived paraffin-embedded and fixed (e.g. formalin-fixed) tissue sample.
General methods for mRNA extraction are well known in the art and are disclosed in standard textbooks of molecular biology, including Ausubel et al., Current Protocols of Molecular Biology, John Wiley and Sons (1997). Methods for RNA extraction from paraffin embedded tissues are disclosed, for example, in Rupp and Locker, Lab Invest. 56:A67 (1987), and De Andrés et al., BioTechniques 18:42044 (1995). In particular, RNA isolation can be performed using a purification kit, buffer set and protease from commercial manufacturers, such as Qiagen, according to the manufacturer's instructions. For example, total RNA can be isolated using Qiagen RNeasy mini-columns. Other commercially available RNA isolation kits include MasterPure™ Complete DNA and RNA Purification Kit (EPICENTRE®, Madison, WI), and Paraffin Block RNA Isolation Kit (Ambion, Inc.). Total RNA from tissue samples can be isolated using RNA Stat-60 (Tel-Test). RNA prepared from heart tissue can be isolated, for example, by cesium chloride density gradient centrifugation.
Typically, the first step in RT-PCR is the reverse transcription of the RNA template into cDNA, followed by its exponential amplification in a PCR reaction. A number of reverse transcriptases may be used, including but not limited to, Avian Myeloblastosis Virus Reverse Transcriptase (AMV-RT), Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV-RT), reverse transcriptase from human T-cell leukemia virus type I (HTLV-I), bovine leukemia virus (BLV), Rous sarcoma virus (RSV), human immunodeficiency virus (HIV) and Thermus thermophilus (Tth). The reverse transcription step is typically primed using specific primers, random hexamers, or oligo-dT primers, depending on the circumstances and the goal of RT-PCR. For example, extracted RNA can be reverse-transcribed using a GeneAmp RNA PCR kit (Perkin Elmer, CA, USA), following the manufacturer's instructions. The derived cDNA can then be used as a template in the subsequent PCR reaction.
Although the PCR step can use a variety of thermostable DNA-dependent DNA polymerases, it typically employs the Taq DNA polymerase, which has a 5'-3' nuclease activity but lacks a 3'-5' proofreading endonuclease activity. Thus, TaqMan® PCR typically utilizes the 5'-nuclease activity of Taq or Tth polymerase to hydrolyze a hybridization probe bound to its target amplicon, but any enzyme with equivalent 5' nuclease activity can be used. Two oligonucleotide primers are used to generate an amplicon typical of a PCR reaction. A third oligonucleotide, or probe, is designed to detect nucleotide sequence located between the two PCR primers. The probe is non-extendible by Taq DNA polymerase enzyme, and is labeled with a reporter fluorescent dye and a quencher fluorescent dye. Any laser-induced emission from the reporter dye is quenched by the quenching dye when the two dyes are located close together as they are on the probe. During the amplification reaction, the Taq DNA polymerase enzyme cleaves the probe in a template-dependent manner. The resultant probe fragments disassociate in solution, and signal from the released reporter dye is free from the quenching effect of the second fluorophore. One molecule of reporter dye is liberated for each new molecule synthesized, and detection of the unquenched reporter dye provides the basis for quantitative interpretation of the data.
TaqMan® RT-PCR can be performed using commercially available equipment, such as, for example, ABI PRISM 7700^™ Sequence Detection System^™ (Perkin-Elmer-Applied Biosystems, Foster City, CA, USA), or Lightcycler (Roche Molecular Biochemicals, Mannheim, Germany). In a preferred embodiment, the 5' nuclease procedure is run on a real-time quantitative PCR device such as the ABI PRISM 7700^™ Sequence Detection System^™. The system consists of a thermocycler, laser, charge-coupled device (CCD), camera and computer. The system amplifies samples in a 96-well format on a thermocycler. During amplification, laser-induced fluorescent signal is collected in real-time through fiber optics cables for all 96 wells, and detected at the CCD. The system includes software for running the instrument and for analyzing the data.
5'-Nuclease assay data are initially expressed as C_t, or the threshold cycle. As discussed above, fluorescence values are recorded during every cycle and represent the amount of product amplified to that point in the amplification reaction. The point when the fluorescent signal is first recorded as statistically significant is the threshold cycle (C_t).
To minimize errors and the effect of sample-to-sample variation, RT-PCR is usually performed using an internal standard. The ideal internal standard is expressed at a constant level among different tissues, and is unaffected by the experimental treatment. RNAs most frequently used to normalize patterns of gene expression are mRNAs for the housekeeping genes glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and β-actin.
A variation of the RT-PCR technique is the real time quantitative PCR, which measures PCR product accumulation through a dual-labeled fluorigenic probe (i.e., TaqMan® probe). Real time PCR is compatible both with quantitative competitive PCR, where internal competitor for each target sequence is used for normalization, and with quantitative comparative PCR using a normalization gene contained within the sample, or a housekeeping gene for RT-PCR. For further details see, e.g. Held et al, Genome Resecarch 6:986-994 (1996).
It is desirable to correct for (normalize away) both differences in the amount of RNA assayed and variability in the quality of the RNA used. Therefore, the assay typically incorporates analysis of the expression of certain reference genes (or "normalizing genes"), including well known housekeeping genes, such as GAPDH, HPRT1, ubiquitin, etc.
Alternatively, normalization can be based on the mean or median signal (C_t) of all of the assayed genes or a large subset thereof (often referred to as a "global normalization" approach). On a gene-by-gene basis, measured normalized amount of a patient heart tissue mRNA may be compared to the amount found in a corresponding normal heart tissue. See M. Cronin, et al., Am. Soc. Investigative Pathology 164:35-42 (2004).

Design of Primers and Probes

Primers and probes (e.g., for use in PCR amplification-based methods) can be designed based upon exon sequence or upon intron sequences present in the gene to be amplified. Accordingly, the first step in the primer/probe design is the delineation of a target exon or intron sequence within the gene of interest. This can be done by publicly available software, such as the DNA BLAT software developed by Kent, W.J., Genome Res. 12(4):656-64 (2002), or by the BLAST software including its variations. Subsequent steps follow well established methods of PCR primer and probe design.
In order to avoid non-specific signals, repetitive sequences within the target sequence of the gene can be masked when designing the primers and probes. This can be easily accomplished by using the Repeat Masker program available on-line through the Baylor College of Medicine, which screens DNA sequences against a library of repetitive elements and returns a query sequence in which the repetitive elements are masked. The masked sequences can then be used to design primer and probe sequences using any commercially or otherwise publicly available primer/probe design packages, such as Primer Express (Applied Biosystems); MGB assay-by-design (Applied Biosystems); Primer3 (Steve Rozen and Helen J. Skaletsky (2000) Primer3 on the WWW for general users and for biologist programmers. In: Krawetz S, Misener S (eds) Bioinformatics Methods and Protocols: Methods in Molecular Biology. Humana Press, Totowa, NJ, pp 365-386).
The factors to be considered in PCR primer design can include primer length, melting temperature (Tm), G/C content, specificity, complementary primer sequences, and 3'-end sequence. In general, optimal PCR primers are 17-30 bases in length, and contain about 20-80% G+C bases, (e.g., about 50-60% G+C bases). Tm's between 50°C and 80 °C, e.g. about 50°C to 70 °C are typically preferred.
For further guidelines for PCR primer and probe design see, Dieffenbach, C.W. et al., "General Concepts for PCR Primer Design" in: PCR Primer, A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1995, pp. 133-155; Innis and Gelfand, "Optimization of PCRs" in: PCR Protocols, A Guide to Methods and Applications, CRC Press, London, 1994, pp. 5-11; and Plasterer, T.N. Primerselect: Primer and probe design. Methods Mol. Biol. 70:520-527 (1997).

Microarrays

Microarray technology may be used to detect differential expression of BIN1 in diseased heart tissue and normal heart tissue. In this method, polynucleotide sequences of interest (including cDNAs and oligonucleotides) are plated, or arrayed, on a microchip substrate. The arrayed sequences are then hybridized with specific DNA probes from cells or tissues of interest. Similar to the RT-PCR method, the source of mRNA typically is total RNA isolated from patient heart tissue, and optionally corresponding normal heart tissue.
Fluorescently labeled cDNA probes may be generated through incorporation of fluorescent nucleotides by reverse transcription of RNA extracted from tissues of interest. Labeled cDNA probes applied to the chip hybridize with specificity to each spot of DNA on the array. After stringent washing to remove non-specifically bound probes, the chip is scanned by confocal laser microscopy or by another detection method, such as a CCD camera. Quantitation of hybridization of each arrayed element allows for assessment of corresponding mRNA abundance. With dual color fluorescence, separately labeled cDNA probes generated from two sources of RNA are hybridized pair wise to the array. The relative abundance of the transcripts from the two sources corresponding to each specified gene is thus determined simultaneously. Microarray methods have been shown to have the sensitivity required to detect rare transcripts, which are expressed at a few copies per cell, and to reproducibly detect at least approximately two-fold differences in the expression levels (Schena et al., Proc. Natl. Acad. Sci. USA 93(2):106-149 (1996)). Microarray analysis can be performed by commercially available equipment, following manufacturer's protocols, such as by using the Affymetrix GenChip technology, or Incyte's microarray technology.
The arrayed oligonucleotides may include oligonucleotides which hybridize to a specific region of BIN1 nucleic acid. In certain embodiments, multiple copies of a first oligonucleotide which specifically hybridizes to a first region of BIN1 nucleic acid are arrayed. In certain embodiments, multiple copies of first and a second oligonucleotide which specifically hybridize to a first and a second region of BIN1 nucleic acid, respectively are arrayed, and so on. In certain embodiments, the BIN1 expression level is determined by mean values of the signal from each of these oligonucleotides. In certain embodiments, the array may also include oligonucleotides which specifically hybridize to nucleic acid of a normalizing gene, such as a housekeeping gene or other genes known not to be significantly differentially expressed in diseased versus normal heart tissue, for example, CaV 1.2.

Immunodetection

Immunohistochemical methods may also be suitable for detecting the expression levels of BIN1. Thus, antibodies or antisera, such as, polyclonal antisera and monoclonal antibodies specific for BIN1 may be used to assess BIN1 expression. The antibodies can be detected by direct labeling of the antibodies themselves, for example, with radioactive labels, fluorescent labels, hapten labels such as biotin, or an enzyme such as horse radish peroxidase or alkaline phosphatase. Alternatively, unlabeled primary antibody is used in conjunction with a labeled secondary antibody, comprising antisera, polyclonal antisera or a monoclonal antibody specific for the primary antibody. In certain examples, BIN1 expression in a heart sample from a patient may be compared to BIN1 expression in a normal heart sample. Immunohistochemistry protocols and kits are well known in the art and are commercially available.
In certain cases, the amount of BIN1 protein present in a heart tissue sample may be determined by a western blot. For example, proteins present in the whole cell lysate from a heart sample may be separated by SDS-PAGE; the separated proteins transferred to a nitrocellulose membrane; BIN1 detected by using an antibody or antiserum specific for BIN1. At least one normalizing protein, for example, Cav1.2 or a housekeeping protein such as GAPDH or β-actin may also be detected simultaneously or in parallel and used to normalize the BIN protein expression levels. In alternative embodiments, BIN1 expression level may be determined by performing a BIN1 immunoprecipitation using an excess of anti-BIN1 antibody, followed by separation of the immunoprecipitate by SDS-PAGE; the separated proteins transferred to a nitrocellulose membrane; and detected by staining the gel, e.g., by Coommassie Blue or silver staining. Immunoprecipitation of a control protein such as GAPDH or ubiquitin may also be carried out either simultaneously or in parallel. Optionally, the same procedure may be carried out on corresponding normal heart tissue.

USE OF BIN1 EXPRESSION LEVELS IN PROGNOSIS OF RISK OF CARDIAC MORTALITY

BIN1 expression can be used as a prognostic marker that is assayed to predict the risk of a poor outcome in a CHF patient. A decrease in BIN1 expression level is positively correlated with an increase in the risk of a poor outcome such as cardiac mortality. BIN1 expression level may be used to calculate an Intrinsic Disease Factor (IDF), which provides a score that increases in value as the risk of a poor outcome such as cardiac mortality increases.

Use of BIN1 Expression Levels

The method can involve assaying BIN1 expression level in a heart tissue sample from a patient diagnosed with CHF. Analysis of BIN1 can involve comparison of the BIN expression level to a reference BIN1 expression level, for example a normal BIN1 expression level or a BIN1 expression level known to be indicative of a low risk of a poor outcome.
The reference BIN1 expression level may be a BIN1 expression level known to be indicative of a low risk of a poor outcome. The reference BIN1 expression level may be the BIN1 expression level in a CHF patient (or an average of BIN1 expression levels of a group of patients) who is known not to have a poor outcome, such as cardiac mortality, LVAD implant, or LVEF that fails to improve. A low BIN1 expression level compared to the reference BIN1 expression level indicates that the patient has an increased likelihood of a poor outcome. In contrast, a BIN1 expression level equal to or greater than the reference value indicates that the patient has a decreased likelihood of a poor outcome.
A normal BIN1 expression level generally refers to a BIN1 expression level in non-failing heart muscle. A normal BIN1 expression level can be determined from BIN1 expression levels of non-failing heart tissue obtained from an individual whose heart function was deduced to be normal from an examination of the gross morphology of the heart, left ventricular ejection fraction, cardiac catheterization, and/or from lack of heart related condition in the individual medical record, and the like.
A low BIN1 expression level compared to a reference BIN expression level indicates that the patient has an increased likelihood of cardiac mortality. In general, at least a 20% reduction in BIN1 expression level compared to a normal BIN expression level indicates that the patient has an increased likelihood of cardiac mortality. Thus, a patient with a 20% reduction, a 30% reduction, a 40% reduction, a 50% reduction, a 75% reduction, or more, in BIN1 expression level compared to a normal BIN expression level has an increased risk of cardiac mortality.
Usually, the BIN1 mRNA or protein level is normalized with reference to an internal control, such as a housekeeping gene or a gene known not to change in CHF, such as CaV1.2. In certain embodiments, the BIN1 mRNA or protein level is normalized with reference to a plurality of internal controls, such as a plurality of housekeeping genes. In general, when a plurality of internal controls is used the mean expression level of these controls is used for normalizing BIN1 expression level.
BIN1 expression levels may be used to predict the risk of a poor outcome over a period of time following the assessment of the BIN1 expression level, for example, the risk of a poor outcome for the CHF patient over the next 1 day-24 months, e.g., 6 months-18 months, such as, over the next 6 months -12 months, or 12 months -18 months..

Intrinsic Disease Factor (IDF)

Intrinsic disease factor (IDF) is calculated as a ratio of control gene(s) expression to BIN1 expression. Accordingly, IDF is represented by the formula: $IDF = [Control / BIN 1]$
wherein "Control" represents an expression value for one or more internal control genes, i.e., a gene(s) which is not significantly differentially expressed in diseased versus normal human heart tissue. Exemplary internal control genes include CaV1.2, and/or housekeeping genes.
IDF_CaV refers to IDF calculated as a ratio of Cav1.2 expression to BIN1 expression and is represented by the formula: ${IDF}_{CaV} = [Cav 1.2 / BIN 1]$
IDF_HK refers to IDF calculated as a ratio of housekeeping (HK) gene(s) expression to BIN1 expression and is represented by the formula: ${IDF}_{HK} = [HK / BIN 1]$
Exemplary housekeeping genes that may be used to determine IDF_HK have been described above. Expression level of a single housekeeping gene may be used to calculate IDF_HK. Alternatively, the mean expression level of two or more housekeeping genes may be used to calculate IDF_HK.
Analysis of IDF can involve comparison of the IDF value to a reference IDF value, e.g., a normal IDF value or an IDF value indicative of a low risk of a poor outcome. A normal IDF value is the ratio of control gene(s) expression to BIN1 expression in non-failing heart tissue. Non-failing heart tissue is obtained from an individual whose heart function was deduced to be normal from an examination of one or more parameters of heart health, such as, the gross morphology of the heart, left ventricular ejection fraction (LVEF), cardiac catheterization, and/or from lack of heart related condition in the individual medical record, etc. For example, a normal heart tissue has a LVEF of 55% or more.
A decrease in BIN1 expression and accordingly increasing IDF values are positively correlated with increasing risk of cardiac mortality, i.e., the higher the IDF, the greater the risk of cardiac mortality. For example, IDF value of greater than about 1.5 times a normal IDF value is indicative of increased risk of cardiac mortality. Thus, an IDF value greater than about 1.5 times, or greater than about 2 times, or greater than about 3 times, or greater than about 4 times, or greater than about 5 times, or more, the normal IDF value is indicative of increased risk of cardiac mortality.
BIN1 expression levels and IDF values for a patient can also be assessed relative to a threshold value. A "threshold value" or "risk threshold value" is a value which can be used to distinguish a relatively high risk of poor outcome from a relatively low risk of a poor outcome. For example, in most cases the threshold value is an approximate value above which risk of a poor outcome is relatively higher, and below which risk of a poor outcome is relatively lower. For example, a normalized BIN1 expression level of about 0.65 represents a threshold value, where cardiac patients having a normalized BIN1 expression level lower than threshold value have a relatively increased risk of a poor outcome, and cardiac patients having a normalized BIN1 expression level greater than or equal to this threshold value have a relatively decreased risk of poor outcome.
BIN1 expression levels and/or IDF value may be used to predict the risk of a poor outcome over a period of time following assessment, for example, the risk of a poor outcome for the CHF patient over the next e.g., 6 months-18 months, such as, over the next 6 months -12 months, or 12 months-18 months..

Clinical Applications

Determination of BIN1 expression level and/or IDF value in heart tissue from a CHF patient may be used to assign a priority level to the patient for receiving a heart transplant, to facilitate assessing response to CHF treatment, and/or to guide modification of a treatment plan.
In general, the lower the BIN1 expression, and/or the higher the IDF value, the higher the likelihood of poor cardiac recovery and hence the patient is assigned a high priority level for receiving a heart transplant. Alternatively, if a CHF patient has a normal or near normal BIN expression level, the expectation is that the heart has a good chance to recover, and the patient might be assigned a low priority level for receiving a heart transplant. In general, the lower the BIN1 expression level and/or the higher the IDF value, the higher is the priority level of the patient for receiving a heart transplant.
Similarly, in patients with only moderately decreased BIN1 expression and/or moderately increased IDF value, there would be increased priority of placing the patient on a left ventricular assist device to help the patient recover. Removal of the left ventricular assist device could be timed with normalization of BIN expression and/or IDF value. A further decrease of BIN1 expression or increase of IDF value despite the left ventricular assist device would indicate listing the patient for heart transplant.
A determination of a risk of cardiac mortality may be used to assess efficacy of CHF treatment and to determine a change to treatment strategy. A CHF patient may be undergoing treatment by, for example, surgery, mechanical assist device, heart transplant, drug therapy, such as, angiotensin-converting enzyme (ACE) inhibitors, angiotensin-receptor blockers (ARBs), beta blockers, diuretics, etc. The efficacy of a CHF treatment may be assessed by assaying for BIN expression level and/or IDF value. In general, a low BIN1 expression, and/or a high IDF value, indicates a high likelihood of a cardiac failure and that the treatment is not efficacious. Such a determination of treatment efficacy may be used to alter the treatment. For example, by classifying the patient at a high priority level for a heart transplant.
On the other hand, a CHF treatment may stabilize the BIN expression level resulting in a normal or near normal expression level. As described in the examples below, as a failing heart recovers, the BIN1 expression level increases. A normal BIN1 expression, and/or a normal IDF value, indicates a decreased likelihood of a cardiac failure and that the treatment is efficacious. This information may be used to classify the patient at a low priority level for receiving a heart transplant, and can indicate other therapies such as an intraortic balloon pump, ventricular assist device, intravenous heart therapy.
A normalized BIN1 expression or IDF value may be used to determine therapy options. For example, a normalized BIN1 expression level less than about 0.65 may be positively correlated to a risk of a poor outcome. A normalized BIN1 expression level of less than about 0.65 may be positively correlated to a poor outcome for the patient in the near future, for example, in the next 1day-24 months, e.g., 1day-7 days, or 1 week-2 weeks, 2 weeks-4 weeks, 1 month-3 months, 3 month-6 months, 6 months-8 months, 8 months-12 months, 12 months-18 months, 18 months-24 months. The poor outcome may be death, LVAD implant, or no improvement in LVEF or even a further deterioration of LVEF. This information may be used to classify the patient at a high priority level for receiving a heart transplant, and can indicate other therapies such as an intraortic balloon pump, ventricular assist device, intravenous heart therapy.
On the other hand, a normalized BIN1 expression level greater than or equal to 0.65 may be negatively correlated to a risk of cardiac mortality. A normalized BIN1 expression level greater than or equal to 0.65 may be positively correlated to absence of poor outcome for the patient in the near future, for example, in the next 1day-24 months, e.g., 1day-7 days, or 1 week-2 weeks, 2 weeks-4 weeks, 1 month-3 months, 3 month-6 months, 6 months-8 months, 8 months-12 months, 12 months-18 months, 18 months-24 months. This information may be used to classify the patient at a low priority level for receiving a heart transplant and other therapies such as an intraortic balloon pump, ventricular assist device, intravenous heart therapy, and can indicate that a drug based therapy may stabilize the heart function of the patient.

Patients

Patients amenable to methods described herein are human patient diagnosed with CHF.
A CHF patient may be a patient diagnosed with end-stage CHF, or acute fulminant myocarditis, or chronic progressive non-ischemic cardiomyopathy, or chronic progressive ischemic cardiomyopathy, or a CHF patient undergoing treatment for CHF, such as, a heart transplant (i.e., post heart-transplant patient), or a patient with refractory CHF, etc.

Kits

The materials for use in the methods of the present disclosure are suited for preparation of kits produced in accordance with well known procedures. The present disclosure thus provides kits comprising agents, which may include gene-specific or gene-selective probes and/or primers, for quantitating the expression of BIN1 for predicting clinical outcome, determining treatment options or predicting response to treatment, etc. Such kits may optionally contain reagents for the extraction of RNA from heart tissue samples and/or reagents for RNA amplification. In addition, the kits may optionally comprise the reagent(s) with an identifying description or label or instructions relating to their use in the methods of the present disclosure. The kits may comprise containers (including microtiter plates suitable for use in an automated implementation of the method), each with one or more of the various reagents (typically in concentrated form) utilized in the methods, including, for example, pre-fabricated microarrays, buffers, the appropriate nucleotide triphosphates (e.g., dATP, dCTP, dGTP and dTTP; or rATP, rCTP, rGTP and UTP), reverse transcriptase, DNA polymerase, RNA polymerase, and one or more probes and primers of the present disclosure. Mathematical algorithms used to estimate or quantify prognostic and/or predictive information are also properly potential components of kits.
The methods provided by the present disclosure may also be automated in whole or in part.

Reports

The methods of the present disclosure are suited for the preparation of reports summarizing the results of assaying the expression level of BIN1. In certain embodiments, a report may include a determination of the risk of cardiac mortality in a CHF patient. A "report," as described herein, is an electronic or tangible document which includes report elements that provide information of interest relating to BIN1 expression level and/or a risk of cardiac mortality. A subject report can be completely or partially electronically generated, e.g., presented on an electronic display (e.g., computer monitor). A report can further include one or more of: 1) information regarding the testing facility; 2) service provider information; 3) patient data; 4) sample data; 5) an interpretive report, which can include various information including: a) indication; b) test data, where test data can include a normalized level of one or more genes of interest, and 6) other features.
The present disclosure thus provides for methods of creating reports and the reports resulting therefrom. The report may include a summary of the expression levels of the RNA transcripts, or the expression products of such RNA transcripts, for certain genes, such as BIN1, in the cells obtained from the patient's heart tissue. The report may include an assessment of risk of cardiac mortality. The report may include a recommendation for treatment modality such as surgery alone or surgery in combination with therapy. The report may be presented in electronic format or on paper. The methods disclosed herein can further include a step of generating or outputting a report, which report can be provided in the form of an electronic medium (e.g., an electronic display on a computer monitor), or in the form of a tangible medium (e.g., a report printed on paper or other tangible medium).
For clarity, it should be noted that the term "user," which is used interchangeably with "client," is meant to refer to a person or entity to whom a report is transmitted, and may be the same person or entity who does one or more of the following: a) collects a sample; b) processes a sample; c) provides a sample or a processed sample; and d) generates data (e.g., level of a response indicator gene product(s); level of a reference gene product(s); normalized level of a response indicator gene product(s)) for use in the risk of cardiac mortality assessment. In some cases, the person(s) or entity(ies) who provides sample collection and/or sample processing and/or data generation, and the person who receives the results and/or report may be different persons, but are both referred to as "users" or "clients" herein to avoid confusion. In certain embodiments, e.g., where the methods are completely executed on a single computer, the user or client provides for data input and review of data output. A "user" can be a health professional (e.g., a clinician, a laboratory technician, a physician (e.g., an cardiologist, surgeon, primary care physician), etc.).
In embodiments where the user only executes a portion of the method, the individual who, after computerized data processing according to the methods of the invention, reviews data output (e.g., results prior to release to provide a complete report, a complete report, or reviews an "incomplete" report and provides for manual intervention and completion of an interpretive report) is referred to herein as a "reviewer." The reviewer may be located at a location remote to the user (e.g., at a service provided separate from a healthcare facility where a user may be located).
Where government regulations or other restrictions apply (e.g., requirements by health, malpractice, or liability insurance), all results, whether generated wholly or partially electronically, are subjected to a quality control routine prior to release to the user.

Computer-Based Systems and Methods

The methods and systems described herein can be implemented in numerous ways. In one embodiment of particular interest, the methods involve use of a communications infrastructure, for example the internet. Several embodiments of the invention are discussed below. It is also to be understood that the present invention may be implemented in various forms of hardware, software, firmware, processors, or a combination thereof. The methods and systems described herein can be implemented as a combination of hardware and software. The software can be implemented as an application program tangibly embodied on a program storage device, or different portions of the software implemented in the user's computing environment (e.g., as an applet) and on the reviewer's computing environment, where the reviewer may be located at a remote site associated (e.g., at a service provider's facility). In some embodiments, the step of using the BIN1 expression level to determine the risk of cardiac mortality in a CHF patient is performed by a computer programmed to execute an algorithm for calculating the risk. In other examples, the subject method includes causing a computer to execute an algorithm for calculating the risk of cardiac mortality in a CHF patient based on the expression level of BIN1.
The application program for executing the algorithms described herein may be uploaded to, and executed by, a machine comprising any suitable architecture. In general, the machine involves a computer platform having hardware such as one or more central processing units (CPU), a random access memory (RAM), and input/output (I/O) interface(s). The computer platform also includes an operating system and microinstruction code. The various processes and functions described herein may either be part of the microinstruction code or part of the application program (or a combination thereof) which is executed via the operating system. In addition, various other peripheral devices may be connected to the computer platform such as an additional data storage device and a printing device.
As a computer system, the system generally includes a processor unit. The processor unit operates to receive information, which can include test data (e.g., level of a response indicator gene product(s); level of a reference gene product(s); normalized level of a response indicator gene product(s)); and may also include other data such as patient data. This information received can be stored at least temporarily in a database, and data analyzed to generate a report as described above.
Part or all of the input and output data can also be sent electronically; certain output data (e.g., reports) can be sent electronically or telephonically (e.g., by facsimile, e.g., using devices such as fax back). Exemplary output receiving devices can include a display element, a printer, a facsimile device and the like. Electronic forms of transmission and/or display can include email, interactive television, and the like. In an embodiment of particular interest, all or a portion of the input data and/or all or a portion of the output data (e.g., usually at least the final report) are maintained on a web server for access, preferably confidential access, with typical browsers. The data may be accessed or sent to health professionals as desired. The input and output data, including all or a portion of the final report, can be used to populate a patient's medical record which may exist in a confidential database at the healthcare facility.
A system for use in the methods described herein generally includes at least one computer processor (e.g., where the method is carried out in its entirety at a single site) or at least two networked computer processors (e.g., where data is to be input by a user (also referred to herein as a "client") and transmitted to a remote site to a second computer processor for analysis, where the first and second computer processors are connected by a network, e.g., via an intranet or internet). The system can also include a user component(s) for input; and a reviewer component(s) for review of data, generated reports, and manual intervention. Additional components of the system can include a server component(s); and a database(s) for storing data (e.g., as in a database of report elements, e.g., interpretive report elements, or a relational database (RDB) which can include data input by the user and data output. The computer processors can be processors that are typically found in personal desktop computers (e.g., IBM, Dell, Macintosh, etc.), portable computers, mainframes, minicomputers, or other computing devices.
The networked client/server architecture can be selected as desired, and can be, for example, a classic two or three tier client server model. A relational database management system (RDMS), either as part of an application server component or as a separate component (RDB machine) provides the interface to the database.
In one example, the architecture is provided as a database-centric client/server architecture, in which the client application generally requests services from the application server which makes requests to the database (or the database server) to populate the report with the various report elements as required, particularly the interpretive report elements, especially the interpretation text and alerts. The server(s) (e.g., either as part of the application server machine or a separate RDB/relational database machine) responds to the client's requests.
The input client components can be complete, stand-alone personal computers offering a full range of power and features to run applications. The client component usually operates under any desired operating system and includes a communication element (e.g., a modem or other hardware for connecting to a network), one or more input devices (e.g., a keyboard, mouse, keypad, or other device used to transfer information or commands), a storage element (e.g., a hard drive or other computer-readable, computer-writable storage medium), and a display element (e.g., a monitor, television, LCD, LED, or other display device that conveys information to the user). The user enters input commands into the computer processor through an input device. Generally, the user interface is a graphical user interface (GUI) written for web browser applications.
The server component(s) can be a personal computer, a minicomputer, or a mainframe and offers data management, information sharing between clients, network administration and security. The application and any databases used can be on the same or different servers.
Other computing arrangements for the client and server(s), including processing on a single machine such as a mainframe, a collection of machines, or other suitable configuration are contemplated. In general, the client and server machines work together to accomplish the processing of the present disclosure.
Where used, the database(s) is usually connected to the database server component and can be any device which will hold data. For example, the database can be a magnetic or optical storing device for a computer (e.g., CDROM, internal hard drive, tape drive). The database can be located remote to the server component (with access via a network, modem, etc.) or locally to the server component.
Where used in the system and methods, the database can be a relational database that is organized and accessed according to relationships between data items. The relational database is generally composed of a plurality of tables (entities). The rows of a table represent records (collections of information about separate items) and the columns represent fields (particular attributes of a record). In its simplest conception, the relational database is a collection of data entries that "relate" to each other through at least one common field.
Additional workstations equipped with computers and printers may be used at point of service to enter data and, in some embodiments, generate appropriate reports, if desired. The computer(s) can have a shortcut (e.g., on the desktop) to launch the application to facilitate initiation of data entry, transmission, analysis, report receipt, etc., as desired.

Computer-readable storage media

The present disclosure also contemplates a computer-readable storage medium (e.g. CD-ROM, memory key, flash memory card, diskette, etc.) having stored thereon a program which, when executed in a computing environment, provides for implementation of algorithms to carry out all or a portion of the calculation of risk of cardiac mortality, as described herein. Where the computer-readable medium contains a complete program for carrying out the methods described herein, the program includes program instructions for collecting, analyzing and generating output, and generally includes computer readable code devices for interacting with a user as described herein, processing that data in conjunction with analytical information, and generating unique printed or electronic media for that user.
Where the storage medium provides a program which provides for implementation of a portion of the methods described herein (e.g., the user-side aspect of the methods (e.g., data input, report receipt capabilities, etc.)), the program provides for transmission of data input by the user (e.g., via the internet, via an intranet, etc.) to a computing environment at a remote site. Processing or completion of processing of the data is carried out at the remote site to generate a report. After review of the report, and completion of any needed manual intervention, to provide a complete report, the complete report is then transmitted back to the user as an electronic document or printed document (e.g., fax or mailed paper report). The storage medium containing a program according to the invention can be packaged with instructions (e.g., for program installation, use, etc.) recorded on a suitable substrate or a web address where such instructions may be obtained. The computer-readable storage medium can also be provided in combination with one or more reagents for carrying out the assaying step of the subject method (e.g., primers, probes, arrays, or other such kit components).

EXAMPLES

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, and temperature is in degrees Centigrade.

Materials And Methods

Plasmids, Cell Culture, and Transfection. Human BIN1 (Isotype 8) cDNA was obtained from Origene. Whole length BIN1-8(1-454aa) and BIN1-BAR*(1-282aa) were then amplified and cloned into pDONR/Zeo (Invitrogen) using Gateway BP cloning to generate entry clones. The genes were subsequently inserted into pDest-eGFP-N1, pDest-mCherry-N1 (converted vectors originally from Clontech), and pcDNA3.2-V5-Dest by Gateway LR cloning. Human CaV1.2 was obtained from Origene. Human β2b and rabbit α2δ1 were provided by Dr. Michael Sangunetti. APC-GFP was provided by Dr. Torsten Wittmann. N-terminal GFP-CaV1.2 was provided by Dr. Kurt Beam, and C-terminal CaV1.2-GFP was described previously (Takahashi et al., 2004). Dominant Negative APC (APC 1-1450aa) was provided by Dr. Ken Kaplan.
HeLa cells and mouse atrial HL-1 cells were cultured in DMEM and Claycomb medium under standard mammalian cell conditions. FuGene 6 (Roche) was used for cDNA transfections in HeLa cells. Lipofectamine (Invitrogen) with plus reagent were used for cDNA transfections in HL-1 cells.
Immunostaining and Electron Microscopy. For all immunocytochemistry, cold methanol fixation (Shaw et al., 2007) was used except for WGA labeling (4% PFA, room temperature) as previously described. For immunohistochemistry, cryosections were fixed in ice-cold acetone for 10 min. For EM tannic acid labeling, cells were fixed with 1.2% glutaraldehyde in the presence of 0.5 1 mg/ml tannic acid. For immunolabeling, mouse cardiomyocyte suspension was fixed in 2% paraformaldehyde with 0.1% glutaraldehyde for 2 hours at room temperature, then washed in 120 mM sodium phosphate buffer and pelleted. Pellets were then infiltrated in PVP sucrose, mounted, and frozen in liquid nitrogen. Immunogold labeling of cryosections was performed as described (Butler et al., 1997). Mouse anti-BIN1 antibody was obtained from Sigma. For CaV1.2, rabbit anti-CaV1.2 from Alamone was used.
Widefield Epifluorescent, TIRF and Spinning Disc Confocal Microscopy. All imaging was performed on a Nikon Eclipse Ti microscope with a 100X 1.49 NA TIRF objective and NIS Elements software. Deconvolution of widefield epifluorescent images was performed using Autoquant software (Media Cybernetics). A laser merge module 5 (Spectral applied research, CA) with DPSS lasers (486, 561, 647nm) was used as a source for TIRF and confocal (Yokogawa, CSU-X1) imaging. Multiple wavelength TIRF was achieved with Dual-View emission splitter (Optical Insights). Time lapse sequence for APC and BIN1 were acquired at a continuous rate of two frames per second with 200ms and 400ms exposure per frame for BIN1 and APC, respectively. High resolution Cool SNAP HQ2 camera and high sensitive Cascade II 512 camera (Photometries) were used for image capture.
Human tissue collection and Cardiomyocytes Isolation. With the approval of the University of California, San Francisco (UCSF) Committee for Human Research, tissue from hearts removed at the time of transplant at UCSF, or from organ donors whose hearts were not transplanted, were obtained. Full informed consent was obtained from all UCSF transplant recipients prior to surgery. The California Transplant Donor Network (CTDN) provided the unused donor hearts and obtained informed consent for their use from the next of kin.
After immediate perfusion with cold cardioplegia, full-thickness samples from left ventricular free wall were cleaned rapidly of all epicardial fat and snap frozen into liquid nitrogen for later protein and mRNA analysis. More sections were embedded in OCT medium and frozen in liquid N2-chilled isopentane for immunohistochemistry. For cardiomyocytes isolation, ventricular free wall samples were cut into ∼1mm3 sections for digestion with pre-warmed collagenase II (2mg/ml, Worthington) at 37°C in calcium free KHB solution (134 mM NaCl, 11 mM Glucose, 10 mM Hepes, 4 mM KCl, 1.2 mM MgSO₄, 1.2 mM Na₂HPO₄, 10 mM BDM, 0.5mg/ml BSA, pH 7.4) (Dipla et al., 1998) with modification of a previously reported method (Beuckelmann et al., 1991). Dissociated cardiomyocytes were allowed to attach to laminin-precoated glass coverslips before fixation for immunocytochemistry.
Isolation and Culture of Adult Mouse Cardiomyocytes. Mouse ventricular myocytes were isolated from male adult C57/Black mouse (8∼12 weeks; Charles River) after dissociation with collagenase II (Worthington, Lakewood, NJ) with previously described method (O'Connell et al., 2007). For surface biotinylation experiments, cardiomyocytes were attached to laminin-precoated culture dishes and cultured in primary cardiomyocyte medium (ScienCell) in 37°C and 5% CO₂ incubator overnight in the presence of 20 µM Dynasore with or without 30 µM Nocodazole.
Surface Biotinylation of CaV1.2. After treatment, the cells were quickly washed and incubated with ice-cold 1mg/ml EZ-linkTM Sulfo-NHS-SS-Biotin (Pierce) for 25 min. After 2 x 5min quenching of unbound biotin with 100 µM glycine, cells were washed and lysed in RIPA buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 2 mM NaF, 200 µM Na₃VO₄) supplemented with Complete Mini protease inhibitor cocktail (Roche). Total protein concentrations were determined and normalized between samples. The lysates were then incubated with prewashed NeutrAvidin coated beads at 4°C overnight. After washes, bound surface proteins were eluted and denatured, separated on NuPage gels (Invitrogen), and probed with rabbit anti-CaV1.2 antibody (Alomone).
Coimmunoprecipitation. Hela cells were cotransfected with human CaV1.2 along with regulatory β2b and a2δ1 subunits and BIN1-V5, harvested, lysed in 1% Triton X-100 Co-IP buffer (50 mM Tris pH 7.5, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 1 mM DTT, 1 mM NaF, 100 µM Na₃VO₄, 1% Triton X-100) supplemented with Complete Mini protease inhibitor cocktail. The lysate was then incubated with either mouse anti V5 antibody (2 µg) or equal amount of non-specific mouse IgG for 2 hours before pull down with rec-protein-G-Sepharose (Invitrogen) for 1 hr. Material bound to washed beads were eluted, denatured, separated, and probed with rabbit antibodies against CaV1.2 (Alomone) or V5 (Sigma).
Quantifying Gene Expression. TaqMan primer/probe sets (5'FAM/3'BHQ; Applied Biosystems) for real-time PCR for human CaV1.2, BIN1, genes were obtained using Primer Express (Applied Biosystems). Total RNA was isolated and purified from left ventricle free wall by TRIzol (Invitrogen) extraction, followed by purification by PureLinkTM RNA mini kit (Invitrogen) and treatment with Turbo DNase (Ambion). First-strand cDNA synthesis was performed using the Superscript First-Strand Synthesis System (BioRad) and oligo-dT primers. Quantitative real-time PCR reactions were performed in a 384-well format using Platinum qPCR mix (Applied Biosystems) and total reaction volumes of 10 µl on an ABI 7900HT (Applied Biosystems). Absolute gene expression was quantified using the method of Dolganov et al. using PP1A, Ubiquitin, EEFA1, PRL13A, and HRPT1 as control genes (Dolganov et al., 2001)(Butler et al., 1997). The primer probes for BIN1 and CaV1.2 were obtained from Applied Biosystems. TaqMan® Gene Expression Assays, Assay ID Hs01120898_ml was used for quantification of BIN1 expression level. This assay detects BIN1 transcript variants 8 and 9. TaqMan® Gene Expression Assays, Assay ID Hs00167681_ml was used for quantification of CaV1.2 expression level. The following probes were used for the housekeeping genes: RPL13A-TMP: CAGAGCGGCCTGGCCTCGCT (SEQ ID NO: 7), Ubiquitin-TMP: TGAGCTTGTTTGTGTCCCTGTGGGTG (SEQ ID NO: 8), EEF1A1-TMP: CACTGGCATTGCCATCCTTACGGG (SEQ ID NO: 9), and PPIA-TMP: ATGGCAAATGCTGGACCCAACACA (SEQ ID NO: 10).
Quantifying Cardiac Ejection Fraction. A transthoracic echocardiogram was performed on the heart which consists of using ultrasound waves from a probe placed on the chest of the patient. The ultrasound waves are used to construct one-dimensional and two-dimensional slices of the heart. Standard volume rendering software on clinical echocardiogram machines (e.g. Acuson) compute left ventricular volume from the slices. The ejection fraction is the difference in volumes during systole and diastole divided by the volume in diastole. Normal ejection fraction is approximately 55-60%.

Example 1. CaV1.2 has an intracellular distribution in failing heart

Cardiac excitation-contraction (EC) coupling, a process essential to each heartbeat, links electrical excitation of the myocyte to its mechanical contraction. EC coupling begins with local calcium entry through CaV1.2 channels that then causes a large release of calcium by the intracellular sarcoplasmic reticulum (Fabiato, 1983). Ryanodine receptors on the sarcoplasmic reticulum (SR) are the local calcium sensors and release channels (Cheng et al., 1993; Inui et al., 1987; Pessah et al., 1985). In ventricular cardiomyocytes, close association of CaV1.2 channels and ryanodine receptors is necessary for efficient calcium-induced-calcium release (CICR) (Bers, 2002). Synchronous CICR occurs at the EC-couplons, where 10-25 L-type calcium channels and 100-200 ryanodine receptors coincide and cooperate with each other (Bers, 2001). In order to locally approximate the SR bound ryanodine receptors, CaV1.2 channels are necessarily enriched on the T-tubule invaginations of the sarcolemma (Scriven et al., 2000). Despite the clear positional necessity of CaV1.2 channels to be enriched at T-tubules, there exists little understanding of the mechanism by which CaV1.2 channels are trafficked and localized to these regions of membrane.
In failing heart, the intracellular calcium transient of ventricular cardiomyocytes has a low amplitude and slow decline (Beuckelmann et al., 1992; Gwathmey et al., 1987; Sipido et al., 1998), resulting in compromised contraction (Harding et al., 1994). Multiple factors downstream of calcium entry through CaV1.2 have been identified in failing muscle that contribute to changes in the calcium transient, including dysfunction in calcium removal (Hasenfuss, 1998; Hasenfuss et al., 1999) and more recently, phosphorylation and dysfunction of the ryandoine release channels (Lehnart et al., 2005; Marx et al., 2000). There have also been reports that dyssynchronous CICR may exist in failing cardiomyocytes and contribute to defective EC-coupling gain in failing heart (Gomez et al., 1997; Litwin et al., 2000).
Reports of CaV1.2 channel expression in dilated cardiomyopathy muscle vary considerably from CaV1.2 being unchanged to decreased (Hullin et al., 1999; Mewes and Ravens, 1994; Schroder et al., 1998). Freshly explanted hearts from transplant recipients with end-stage non-ischemic dilated cardiomyopathy and non-failing hearts from organ donors that could not be used for transplantation were obtained. After immediate perfusion with cardioplegia, ventricular sections were frozen and ventricular cardiomyocytes were also isolated. Immunohistochemistry of cryosections shows staining of CaV1.2 and a marker for T-tubules, the T-tubule formation protein BIN1) (Lee et al., 2002). In non-failing myocardium, CaV1.2 distribution is well organized along T-tubules marked with BIN1. However, in failing myocardium, T-tubular BIN1 distribution is disrupted along with CaV1.2. Interestingly, data shown in Figure 1A indicate that CaV1.2 expression is unchanged both at mRNA and protein levels in failing myocardium. These data indicate that the cellular distribution of CaV1.2, rather than expression level, is altered in failing myocardium.
To better understand the cellular distribution of CaV1.2, isolated human cardiomyocytes were stained and imaged at Z-depth increments of 0.1 µm with a spinning disc confocal microscope. The Z-axis cross sections of cardiomyocytes were generated from the original z-stack and shown in the side images. Fluorescent intensity at cell peripheral within 2 µm of cell edges were quantified and normalized to total cellular fluorescent intensity and presented in the bottom right panel. Peripheral fluorescent signal is significantly reduced in failing cardiomyocytes. Three dimensional volume views and Z-axis cross sections are shown in Figure 1B. As seen in the subsection of a non-failing cardiomyocyte (left), CaV1.2 is enriched at T-tubules, whereas in diseased cardiomyocytes CaV1.2 has an intracellular distribution and the distinctive T-tubular invaginations are lost. Peripheral CaV1.2 quantification indicates that the peripheral proportion of CaV1.2 (within 2 µm from cell edges concentrated of T-Tubules) is significantly reduced in failing cardiomyocytes. CaV1.2 internalization in failing cardiomyocytes can also be seen in full 360 degree rotations. Given that CaV1.2 protein and mRNA are unchanged (Figure 1A), internalization of L-type calcium channels in failing cardiomyocytes indicates improper post-Golgi trafficking of the channel.

Example 2. BIN1 Targets CaV1.2 to T-Tubules

The mechanism of CaV1.2 targeting to T-tubule membranes was explored. Co-staining of BIN1 and CaV1.2 in both isolated non-failing human cardiomyocytes and mouse cardiomyocytes indicates that BIN1 outlines T-tubules and CaV1.2 is enriched along such invaginations and colocalizes with BIN1. For higher resolution imaging, transmission electron microscopy with dual immunogold labeling was used to identify CaV1.2 and BIN1 on T-tubule ultrastructures in adult mouse cardiomyocytes. A representative image in Figure 2A indicates that BIN1 (small 10nm dots) and CaV1.2 (large 15nm dots) are enriched and cluster within 10-50 nm of each other, occurring at T-tubular membrane structures.
Given that CaV1.2 is closely associated with the tubulogenesis protein BIN1 at T-tubules, it is possible that BIN1 specifically attracts delivery of CaV1.2 to T-tubules. This possibility was tested using the reductionist approach of studying the association between BIN1 and CaV1.2 in an atrial myocyte cell type HL-1 cells that do not have the capability of forming T-tubules but do express endogenous CaV1.2 (*), and non-myocyte Hela cells that have neither T-tubules nor CaV1.2. In both cell lines, exogenous BIN1 is able to induce nascent T-tubule-like membrane invagination. In HL-1 cells, fixed cell immunocytochemistry indicates that endogenous CaV1.2 colocalizes with exogenous BIN1. Also, BIN1 forms linear tracks which corresponds to T-tubule-like structures (Lee et al., 2002).
Similar colocalization results are obtained with non-myocyte HeLa cells transfected with exogenous BIN1 and CaV1.2. Surface expression of CaV1.2 was evaluated using total internal reflection fluorescence (TIRF) microscopy which limits the imaging depth to within 50-100 nm of the coverslip. Using CaV1.2 and BIN1 tagged with spectrally distinct fluorophores, a brief time lapse capture was performed. The results indicate that, at the membrane surface, BIN1 induced structures attract surface CaV1.2 thereby causing local enrichment of calcium channel. The HeLa cell data are intriguing, indicating that in the absence of other myocyte structures as well as the absence of endogenous CaV1.2, membrane anchoring protein BIN1 and CaV1.2 will still congregate when overexpressed. Close biochemical association between BIN1 and CaV1.2 in Hela cells is further supported by positive co-immunoprecipitation of V5 tagged BIN1 and probed for CaV1.2. (Figure 2B).

Example 3. CaV1.2 is Targeted to BIN1, not T-Tubules

It was determined whether it is BIN1 protein or T-tubule structures that is sufficient for CaV1.2 targeting. Wild-type BIN1 (BIN1-WT, 1-454aa) has an N-terminal BAR domain followed by 15 amino acids (aa) encoded by exon 10 which is required for phospholipid binding and T-tubule formation, a coil-coiled linkage domain, and a C-terminal SH3 domain for protein-protein interaction (Figure 3A) (Lee et al., 2002; Nicot et al., 2007). A previously published C-terminal truncated BIN1-BAR* (1-282aa, BAR+Exon10), which retains the ability to induce membrane invagination was created (Lee et al., 2002). BIN1-BAR* cannot attract endogenous CaV1.2 to the nascent T-tubule structures such as those in HL-1 cells. In an HL-1 cell transfected with BIN1-WT, endogenous CaV1.2 is distributed along BIN1 structures. In contrast, in cells transfected with BIN1-BAR*, CaV1.2 shows poor colocalization with the BIN1 structures. The effect of BIN1-WT and BIN1-BAR* on CaV1.2 surface targeting was further tested by a biochemical surface biotinylation assay. As the data in Figure 3B indicate, unlike BIN1-BAR*, BIN1-WT induces surface expression of CaV1.2. The assay indicates that T-tubule targeting of CaV1.2 requires full length BIN1; T-tubule structures induced by BIN1-BAR* are not sufficient to attract CaV1.2.

Example 4. Failing Cardiomyocytes Express Less BIN1 and Have Shallow T-Tubules

Since BIN1 generates T-tubules and targets CaV1.2 to T-tubules (Figure 3), it was determined whether the internalization of CaV1.2 (Figure 1) in failing cardiomyocytes is the result of abnormal BIN1 expression. BIN1 mRNA and protein expression was measured in failing human hearts by quantitative RT-PCR and western blot, respectively. Compared to non-failing heart tissue, failing myocardium has a significantly lower message and protein level of BIN1 (Figure 4A). Isolated human cardiomyocytes were also used to evaluate BIN1 expression at the cellular level. Tubular BIN1 expression and the T-tubule depth indicated by BIN1 staining in the cross sections were quantified and summarized (Figure 4B). As indicated in the cross-section, human failing cardiomyocytes have much lower BIN1 expression along T-tubules. Quantification of BIN1 signal in T-tubules indicates that failing cardiomyocytes have less BIN1 signal and shallower T-tubules. Three dimensional reconstruction of membrane labeling with wheat germ agglutinin (WGA) confirms disruption of the T-tubule network in failing cardiomyocytes. Cross-sections indicate that failing cardiomyocytes have shallower and diminished T-tubules (Figure 4B, bottom row). In summary, data from Figures 1-4 indicates that BIN1 induces T-tubule formation and targets CaV1.2 to T-tubule membranes; in failing cardiomyocytes, BIN1 is decreased, resulting in shallow T-tubules and reduced membrane associated CaV1.2.

Example 5. CaV1.2 Targeting Involves APC-Tipped Microtubules

To evaluate the role of microtubules in trafficking of CaV1.2, endogenous L-type calcium channels (CaV1.2) were co-stained with α-tubulin in fixed atrial HL-1 cells and examined with high resolution imaging (with deconvolution post-processing). Results indicate that CaV1.2 is concentrated in the perinuclear Golgi and distributes along the microtubule network. To confirm that the CaV1.2 channels associated with microtubules are being trafficked to the membrane (i.e. forward trafficking), live HL-1 cells and adult cardiomyocytes were exposed to the microtubule disruptor nocodazole in the presence Dynasore, which is a dynamin GTPase inhibitor that blocks endocytosis (Macia et al., 2006). Expression of surface CaV1.2 was assayed by surface biotinylation. Results from the adult cardiomyocytes in Figure 5 indicate that microtubule disruption results in less forward trafficking of CaV1.2 to the membrane. Similar results were obtained for surface expression of endogenous CaV1.2 in HL-1 cells.
Next, it was evaluated if a +TIP protein was associated with CaV1.2 delivery to BIN1 at T-tubules. EB1 distribution in cardiomyocytes was found to be concentrated at the intercalated disc rather than T-tubules. Whereas EB1 is typically associated with cell-cell border regions (Shaw et al., 2007), the +TIP protein APC has a different cellular distribution (Barth et al., 2002) and is more commonly associated with extending membrane of migrating cells (Etienne-Manneville and Hall, 2003; Neufeld and White, 1997). More importantly, APC is involved in epithelia tubulogenesis (Pollack et al., 1997). Therefore, the distribution of APC in cardiomyocytes was explored. Triple staining of α-tubulin, APC, and CaV1.2 in mouse cardiomyocytes revealed that APC resides in the regions of T-tubules and Z-lines, and that APC tipped microtubules frequently cluster in regions of CaV1.2 enrichment. To test whether APC-tipped microtubules are involved in delivery of endogenous CaV1.2, a dominant negative form of APC (Green et al., 2005) was used in atrial HL-1 cells. Surface biotinylation indicated that interfering with APC function reduces surface expression of CaV1.2 (Figure 6A). Thus, APC has a role in delivering CaV1.2 channels to the sarcolemma.
Given that EB1-tipped microtubules can be anchored to adherens junctions structures (Ligon and Holzbaur, 2007), facilitating the delivery of ion channels (Shaw et al., 2007), live cell imaging was used to evaluate whether APC tipped microtubules could be anchored by BIN1. HeLa cells were transfected with BIN1-mCherry and APC-GFP and imaged for 120 seconds with both mCherry and GFP signals captured alternatively at an average frame rate of 2.5 seconds. APC-tipped microtubules typically concentrate at the leading edge regions of the cell. Manual traces of the path of six individual APC-tipped microtubules during the imaging interval were obtained. When APC is in the vicinity of BIN1, APC-tipped microtubules linger around the BIN1 structure. In contrast, at cell edges absent of BIN1 structures, APC-tipped microtubules travel a longer distance at a faster velocity, without being anchored. The full data of APC particle path length and velocity (Figure 6B) indicate that APC anchors microtubules to BIN1 facilitating delivery of CaV1.2 to BIN1 containing membrane.

Example 6. BIN1 expression level in heart on LVAD

Heart from an end stage dilated cardiomyopathy patients who had received a left ventricular assist device (LVAD) at LV Apex was analyzed to determine BIN1 expression level. BIN1 expression level was determined in heart muscle from the LV-FW and the LV-APEX of the patient. This patient had been on the LVAD for about six months. BIN1 expression level was also determined in corresponding tissue from individuals who died for reasons other than heart disease. The BIN 1 expression levels are shown in Figure 7. Figure 7 shows that non-failing heart from individuals who died for reasons other than heart disease exhibit comparable BIN1 expression levels in left ventricle free wall (LVFW) and LV Apex (A), while hearts from end stage dilated cardiomyopathy patients who received LVAD at LV Apex show a recovery of BIN1 expression in the LV apex heart tissue (compare to LV-FW) (B).

Example 7. IDF _CaV1.2 and/or IDF _HK values and heart health

IDF_CaV is a ratio of CaV1.2 mRNA expression level to BIN1 mRNA expression level. IDF_HK is a ratio of HPRT1 (or another housekeeping gene) expression level to BIN1 mRNA expression level. IDF_CaV and IDF_HK values were determined for failing and non-failing heart. Results of the analysis of the IDF_CaV values is shown in Figure 8. Figure 8 shows that cardiac IDF_CaV is significantly higher in hearts with end-stage dilated cardiomyopathy. Figure 8 (A) shows IDF_CaV for non-failing heart from individuals who died for reasons other than heart disease (filled circles) and for failing hearts from end stage dilated cardiomyopathy patients (filled squares). Figure 8 (B) shows that cardiac IDF_CaV correlates with left ventricular ejection fraction (LVEF). IDF_CaV for non-failing heart from normal control individuals who died for reasons other than heart disease (filled squares) and for failing hearts from end-stage dilated cardiomyopathy patients (filled circles) are depicted.
Results of the analysis of the IDF_HK values (measured as a ratio of a HPRT1 mRNA to BIN1 mRNA) is shown in Figure 9. Figure 9 shows that cardiac IDF_HK is significantly higher in hearts with end-stage dilated cardiomyopathy. Figure 9 (A) shows IDF_HK values for non-failing heart from normal control individuals who died for reasons other than heart disease (filled circles) and for failing hearts from end-stage dilated cardiomyopathy patients (filled squares). Figure 9 (B) shows that cardiac IDF_HK correlated to left ventricular ejection fraction (LVEF). IDF_HK for non-failing heart from normal control individuals who died for reasons other than heart disease (filled squares) and for failing hearts from end-stage dilated cardiomyopathy patients (filled circles) are depicted.

Example 8: BIN1 Expression Correlates to Cardiac Output in Heart Failure Patients

Endomyocardial biopsies were obtained from the right ventricular septum from fourteen patients with heart failure of unknown etiology during clinical evaluation. These patients exhibited idiopathic non-infiltrative cardiomyopathy and reduced cardiac output (lower than 4.5 L/min). The heart biopsies were analyzed by quantitative immunohistochemistry for BIN1 and, for sample normalization, Cav1.2 content. For each biopsy sample, individual cardiomyocyte cross sections were outlined for measurement of cellular levels of BIN1 and Cav1.2 (at least ten cardiomyocytes per sample). As seen in Figure 10, there is a strong correlation between normalized BIN1 levels and cardiac output.

Example 9: BIN1 Expression Predicts Outcome in Heart Failure Patients

The fourteen patients, from whom endomyocardial biopsies were obtained and normalized BIN1 levels determined (see Example 8 above), were followed-up after 6-18 months to ascertain whether the normalized BIN1 levels were predictive of outcome.

Death, LVAD implant, or LVEF less than 25% was considered as a poor outcome. The results are presented in Table 1 below:

Table 1

ID	BIN1/Cav1.2	Bad Outcome?	Note
51	0.49	yes (LVEF 10%)
53	0.59	yes (LVEF 20%)
87	0.60	yes (LVAD))
68	0.61	no		Test Cutoff
77	0.64	yes (died)		(Bin1/Ca <0.65)
91	0.66	no
61	0.68	no
52	0.75	no
76	0.84	yes (LVEF 20%)
80	0.85	no
55	0.86	no
86	0.89	no
75	0.93	yes (LVAD)	Giant Cell Myocarditis
73	1.12	no

Low normalized BIN1 expression level positively correlated with a Poor outcome (Table 1). Table 1 shows that patients with low BIN1 expression levels generally did not respond to therapy and had a poor outcome, i.e., death, no improvement in LVEF, or needed a LVAD implant. On the other hand, patients with high BIN1 expression levels generally did not show a Poor outcome.
Based on the follow-up data, a normalized BIN1 value of less than 0.65 was determined to be the cut-off value. Normalized BIN1 expression level less than 0.65 indicated a positive test for poor outcome and normalized BIN1 expression level equal to or more than 0.65 indicated a negative test for poor outcome.
Using the 2x2 box in Table 2, sensitivity is obtained by dividing true test positives (4) by the sum of true positives and false negatives (4+2=6). Specificity is obtained by dividing true test negatives (7) by the sum of true negative and false positive (7+1=8). Positive predictive value is obtained by dividing true test positives (4) by the sum of true positive and false positive (4+1=5). Negative predictive value is obtained by dividing true test negatives (7) by the sum of true negative and false negative (7+2=9).
As seen in Table 2, a positive test is 67% predictive (normalized BIN1 level <0.65) of a poor outcome as provided above. A negative test (normalized BIN1 level ≥0.65) has a specificity of 88% (a negative test translates to an 88% chance that heart will not have a poor outcome in the at least next 6-18 months). The positive predictive value (PPV) of BIN1 expression level for a poor outcome was 80% and the negative predictive value (NPV) of BIN1 expression level for absence of poor outcome was 78%.

Example 10. Assessing the health of heart on mechanical assist device

A patient with end-stage dilated cardiomyopathy has a LVAD implanted to aid with heart pump function. It is clinically difficult to determine whether his heart is recovering from therapy and the LVAD can be safely removed, or whether the heart continues to deteriorate. The patient, who may be either an inpatient on the cardiac service or an outpatient under the care of a cardiac team, is scheduled for a ventricular biopsy in the cardiac catheterization laboratory. After a six hour fast the patient is brought to the catheterization laboratory and provided with mild conscious sedation as well as local anesthetic (lidocaine) to an access point of either in the internal jugular vein (neck) or femoral vein (groin). A cardiac biotome is introduced and advanced to the right ventricle under fluoroscopic guidance. The biotome is used to obtain a single biopsy of the right ventricle which is immediately flash frozen in liquid nitrogen. After 2-4 hours of monitoring, the patient is either returned to his room or discharged home from the catheterization lab. Alternatively, the cardiac biotome can be advanced to the left ventricle and then used to obtain a biopsy of the left ventricle.
The tissue sample is either stored at -80 degree Celsius freezer or immediately processed. Tissue is processed using standard laboratory techniques for RNA extraction and gene expression is determined by quantitative RTPCR (qRTPCR). Expression levels of cardiac BIN1 and/or BIN1 and cardiac CaV1.2, and/or BIN1 and HPRT1 is measured using qRTPCR.
An increased expression of cardiac BIN1 in LV-Apex compared to the LV-FW indicates that the patient is responding to LVAD treatment. Once subsequent determination of BIN1 expression levels indicate that the BIN1 expression level has stabilized, the patient is diagnosed as treated and the LVAD is removed.
As an additional indicator of heart health, the Intrinsic Disease Factor (IDF) is also determined and is used for clinical decision making. IDF_CaV is a ratio of CaV mRNA expression level to BIN1 mRNA expression level. IDF_HK is a ratio of HPRT1 (or another housekeeping gene) expression level to BIN1 mRNA expression level. A severely elevated IDF (for instance IDF_CaV of greater than 30 (Figure 7) or IDF_HK of great than 15 (Figure 8) or, any similar normalized IDF greater than 1.5 times control IDF) indicates that the heart has severe heart failure. A patient with a severely elevated IDF is diagnosed as requiring long-term LVAD therapy. In subsequent determination of IDF, absence an improvement from LVAD therapy as determined by a high IDF, a heart-transplant is advised. In contrast, a patient with normal or mildly elevated IDF (normalized IDF less than 1.5 times control IDF).will indicate potential recovery of heart function and, ultimately, successful removal of the LVAD.
BIN1 expression is measured in biopsies of heart tissue at regular intervals, of about one month, to establish a trend to assess the rate of cardiac recovery (or deterioration).
Thus, BIN1 expression levels and/or IDF can distinguish between hearts that can recover by LVAD therapy and hearts that cannot.

Example 11: Analysis of BIN1 expression levels in end stage CHF patients

Prognostication using currently available methods is extremely difficult for patients with end-stage congestive heart failure. Because BIN1 expression level as well as IDF quantifies the extent of disease related changes in individual cardiomyocytes, they provide prognostic data on subsets of severe heart failure patients. Specific subsets include acute fulminant myocarditis, chronic progressive non-ischemic cardiomyopathy, chronic progressive ischemic cardiomyopathy, and post heart-transplant patients.
Endomyocardial tissue biopsies from these four subsets of patients are obtained to conduct retrospective studies. For each patient, data on cardiac diagnosis, left ventricular ejection fraction, age, sex, and cardiac outcome, such as, survival time post biopsy is obtained. Heart tissue from patients about whom the clinical data are incomplete is not included in the study.
Tissue obtained by biopsy is flash frozen in liquid nitrogen and qRTPCR is performed as described above. Archived heart tissue preserved as formalin-fixed paraffin-embedded (FFPE) sample is either analyzed by qRTPCR or by immunohistochemistry. For immunohistochemistry primary antibodies against BIN1 and/or BIN1 and CaV1.2 and/or BIN1 and a housekeeping protein are used. A reduction in gene expression corresponds to reduction in fluorescence obtained with immunohistochemistry. An IDF is also obtained with quantitative immunohistochemistry of protein levels in a manner similar to the ratios obtained with the qRTPCR data, as described above.
For each of the four datasets as well as combinations of the datasets, a survival curve, such as a Kaplan-Meyer curve is derived. Survival in the patient groups is tracked and is compared to the BIN1 expression level or IDF at the time of biopsy. Patients with elevated IDF (e.g. initial value greater than 1.5 of controls without cardiac disease) have lower expected survival than patients with IDF of less than 1.5 of controls without cardiac disease.

Example 12: Using BIN1 expression levels in diagnosing heart health

Patients with acute fulminant myocarditis have a strong indication for endomyocardial biopsy (to rule out giant cell myocarditis and eosiniphilic myocarditis). Similarly, patients with end stage ischemic and non-ischemic cardiomyopathy also frequently undergo biopsy as part of the transplantation work-up. Patients who receive a ventricular assist device such as a LVAD, a right ventricular assist device (RVAD), or a biventricular assist device (BiVAD), generate a large biopsy from the ventricular core removed to insert the device. Furthermore, biopsy is frequently obtained from patients post heart transplant as either an annual screen for pre-clinical rejection or during acute decompensation periods to rule out acute rejection.
The Intrinsic Disease Factor (IDF) is obtained from the ratio of an internal control to BIN1, both assayed by qRTPCR. Internal controls are a standard set of housekeeping genes (HK), such as HPRT1 or CaV1.2. HK genes and CaV1.2 gene expression levels are not affected in heart failure.
BIN1 expression levels and/or BIN1 derived IDF is a clinical snapshot of myocardial health. Severely high IDF (estimated at greater than 1.5 of control IDF) indicates poor myocardial health with prognostic implications of low recovery. As discussed in Example 9 above, IDF prognosticates mortality based on the initial value (IDF greater than 1.5 of control IDF corresponds to high mortality). Institution of therapy such a cardiovascular drugs and mechanical assist devices can be followed by weekly or monthly biopsies to track progression of recovery. IDF is also useful in post-transplant patients requiring screening for rejection. Current biopsy examination has relatively poor sensitive and specificity because the biopsy samples is studied for signs of inflammation and immune cells which can both be present in a healthy heart and absent in a particular sample of a heart undergoing rejection. By assaying the cell biological processes of the cardiomyocytes themselves, BIN expression level and BIN1 derived IDF improve both sensitive and specificity of detection of transplant rejection.

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SEQUENCE LISTING

<110> The Regents of the University of California Shaw, Robin Hong, Ting-Ting Smyth, James
<120> Bin1 as a Prognostic Marker in
Cardiovascular Disease
<130> UCSF-411
<150> 61/172,608 <151> 2009-04-24
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Claims

A method for determining a risk of a poor outcome in a congestive heart failure patient, the method comprising:
assaying a Bridging integrator 1 (BIN1) expression level in a heart tissue sample obtained from the patient, wherein the patient is a human patient; and

using the BIN1 expression level to determine the risk of a poor outcome in the patient, wherein a decreased BIN1 expression level relative to a reference BIN1 expression level is positively correlated to an increased risk of a poor outcome.
The method of claim 1, wherein the assaying comprises measuring the level of BIN1 mRNA.
The method of claim 1, wherein the assaying comprises measuring the level of BIN1 protein.
The method of claim 1, comprising assaying an internal control gene expression level and normalizing the BIN1 expression level using the assayed internal control gene expression level.
The method of claim 4, comprising assaying a CaV1.2 expression level and normalizing the BIN1 expression level using the assayed CaV1.2 expression level.
A method for determining a risk of a poor outcome in a congestive heart failure patient, the method comprising:
assaying expression levels of Bridging integrator 1 (BIN1) and an internal control gene in a heart tissue sample obtained from the patient, wherein the patient is a human patient;

determining an Intrinsic Disease Factor (IDF) by calculating a ratio of the internal control gene expression level to the BIN1 expression level; and

using the IDF to determine the risk of a poor outcome in the patient, wherein an increased IDF relative to a reference IDF is positively correlated to an increased risk of a poor outcome.
The method of claim 6, wherein the internal control gene is a housekeeping gene.
The method of claim 6, wherein the internal control gene is CaV1.2.
The method of claim 6, wherein the assaying comprises measuring the levels of BIN1 and the internal control gene mRNA.
The method of any one of claims 1 to 9, wherein the congestive heart failure patient has received a heart transplant and the heart tissue sample is from the transplanted heart.
The method of any one of claims 1 to 9, wherein the heart of the congestive heart failure (CHF) patient is connected to a mechanical assist device.
The method of any one of claims 1 to 9, wherein the patient is undergoing treatment for CHF.
The method of claim 10, wherein the patient is undergoing immunosuppression therapy.
The method of any one of claims 1 to 9, wherein the poor outcome is cardiac mortality.
The method of any one of claims 1 to 9, wherein the poor outcome is a failure of left ventricular ejection fraction (LVEF) to improve, the need for mechanical device support, or the need for a heart transplant.